Stimulation of T lymphocytes by antigen or anti-T cell receptor (TcR) antibodies causes rapid phosphorylation of several cellular proteins on tyrosine residues. Tyrosine phosphorylation signals are prerequisite for all subsequent events of T cell activation, including mobilization of intracellular calcium and the secretion of lymphokines such as interleukin-2 (IL-2). However, the TcR, the TcR-associated CD3 complex and .zeta. chain are devoid of intrinsic catalytic properties. This suggests that TcR-induced tyrosine phosphorylation is mediated through recruitment of various cytoplasmic protein tyrosine kinases (PTKs). Indeed, accumulating evidence shows that two members of the src family of protein tyrosine kinases, p56.sup.lck and p59.sup.fyn, as well as the Syk-related kinase ZAP-70, are involved in the initiation of the TcR-induced signaling cascade. For reviews on TcR signal transduction, see Perlmutter, R. M. et al. (1993) Annu. Rev. Immunol. 11:451-499 and Chan, A. C. et al. (1994) Annu. Rev. Immunol. 12:555-592.
The protein tyrosine kinase p59.sup.fyn (also referred to as Fyn) is a 59 kilodalton intracellular protein expressed predominantly in neuronal and hematopoietic cells, including T lymphocytes. A number of experiments suggest a role for Fyn in TcR-mediated T cell responses. Using sensitive in vitro kinase assays and mild detergents for solubilization, an association between Fyn and the TcR complex has been demonstrated (see e.g., Samelson, L. E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:4358-4362; Sarosi, G. P. et al. (1992) Int. Immunol. 4:1211-1217; Gassman, M. et. al. (1992) Eur. J Immunol. 22:283-286). Moreover, ligation of the TcR has been reported to activate the kinase activity of Fyn in various human T cell lines (Tsygankov, A. Y. et al. (1992) J. Biol. Chem. 267:18259-18262; da Silva, A. J. et al. (1992) Mol. Immunol. 29:1417-1425; Burkhardt, A. L. et al. (1994) J. Biol. Chem. 269:23642-23647). Still further, mature T cell lines that underexpress Fyn have been shown to exhibit TcR signaling defects, namely a reduction of the phosphorylation of proteins on tyrosine residues or a lower mobilization of calcium following TcR/CD3 activation, compared to wild-type cells (Lee, S.-K. et al. (1994) Int. Immunol. 6:1621-1627; Rigley, K. et al. (1995) J. Immunol 154:1136-1145). A role for Fyn in TcR signaling has also been suggested by genetic studies. For example, thymocytes from transgenic mice expressing high levels of Fyn are hyper-responsive to stimulation via the TcR (Cooke, M. P. et al. (1991) Cell 65:281-292). Furthermore, disruption of the fyn gene in mice by homologous recombination results in defective signaling in single positive thymocytes (Appleby, M. W. et al. (1992) Cell 70:751-763; Stein, P. L. et al. (1992) Cell 70:741-750).
Structurally, Fyn is a member of the src family of PTKs, exhibiting a src homology 2 (SH2) domain, responsible for association with phosphotyrosine-containing peptides, and a src homology 3 (SH3) domain, thought to mediate binding to proline-rich peptides. A limited number of proteins have been shown to interact with Fyn through its SH2 and/or SH3 domain upon T cell activation. These proteins include Irs-1 (Sun, X. J. et al. (1996) J. Biol. Chem. 271:10583-10587), Sek, an Eph family receptor tyrosine kinase (Ellis, C. et al. (1996) Oncogene 12:1727-1736), an embryonal protein Efs (Ishino, M. et al. (1995) Oncogene 11:2331-2338), the phosphatidyl inositol-3-kinase (Prasad K. V. et al. (1993) Proc. Natl. Acad. Sci. USA 90:7366-7370; Pleiman, C. M. et al. (1994) Science 263:1609-1612) and the HS1 protein (Baumann, G., et al. (1994) Eur. J. Immunol. 24:1799-1807), as well as polypeptides of 62, 82 and 120/130 kDa of unknown identity (da Silva, A. J. et al. (1993) J. Exp. Med. 178:2107-2113). Identification and characterization of proteins capable of interacting with Fyn in T cells is important for understanding the role of Fyn in T cell activation and, accordingly, for designing approaches to modulate this process.